The HIV-1/AIDS epidemic continues to spread at an alarming rate world-wide, despite considerable efforts to control it. Central nervous system (CNS) infection by HIV-1 is common and occurs early after systemic infection. Sequence analysis has indicated that the virus may evolve independently in the CNS, probably resulting in adaptation to replication in microglia and macrophages within the brain. However, the potential mechanisms and phenotypic consequences of such adaptation are not well understood. HIV-1 enters cells through the sequential interactions of the viral envelope surface glycoprotein subunit gp120 with the main host cell receptor CD4, and a co-receptor (mainly CCR5 and/or CXCR4). It was previously reported that a primary, peripheral HIV-1 isolate adapted in vitro to replicate in microglial cells acquired increased fusion capacity concurrently with lower CD4 dependence and altered envelope conformation and sensitivity to antibodies and entry inhibitors. Due to the low levels of CD4 in microglial cells and macrophages, and the relative immunological isolation of the CNS, it is hypothesized that viruses containing envelope glycoproteins with similar phenotypes (low CD4 dependence, high fusogenicity and altered sensitivity to entry inhibitors) will arise in vivo as a consequence of viral adaptation to replication in microglial cells and brain macrophages. Preliminary studies have shown that envelope genes amplified from brain tissue feature the above phenotype (including significantly increased sensitivity to a novel allosteric inhibitor of gp120, HNG-105), while those from peripheral tissues do not. However, it is not clear whether viral isolates recovered from brain tissue have this phenotype, as well as the potential role of these phenotypic features in neurotoxicity and neurovirulence. Therefore, the goals of this application are: (i) to determine the phenotypes (CD4 dependence, fusogenicity, cell tropism) of viral isolates recovered from brain and spleen, and of authentic envelopes generated by single genome amplification, from HIV patients with various degrees of neurocognitive impairment; (ii) to define the relationship between these phenotypes and altered sensitivity to two selected, prototypic entry inhibitors (BMS- 378806 and HNG-105) and to identify viral determinants for the phenotypic differences; and (iii) to determine neurotoxicity of viral isolates and envelopes and to define the potential role of these entry inhibitors in decreasing neurotoxicity. Altogether, these studies will provide a functional analysis of the in vivo evolution of HIV-1 in the CNS, complementing previous genetic studies, and will help define a critical correlation between low CD4 dependence, fusogenicity and macrophage tropism, with in vitro neurotoxicity and neurovirulence in vivo, and with an altered sensitivity to specific entry inhibitors, which will potentially allow more targeted approaches for the particular inhibition of replication o viruses present in the CNS of HIV-1-infected patients.